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The actinomycete genus Rhodococcus is known for its diverse biosynthetic enzymes, with potential in pollutant degradation, chemical biocatalysis, and natural product exploration. Comparative genomics have analyzed the distribution patterns of non-ribosomal peptide synthetases (NRPSs) in Rhodococcus . The diversity and specificity of its secondary metabolism offer valuable insights for exploring natural products, yet remain understudied. In the present study, we analyzed the distribution patterns of biosynthetic gene clusters (BGCs) in the most comprehensive Rhodococcus genome data to date. The results show that 86.5% of the gene cluster families (GCFs) are only distributed in a specific phylogenomic-clade of Rhodococcus , with the most predominant types of gene clusters being NRPS and ribosomally synthesized and post-translationally modified peptides (RiPPs). In-depth mining of RiPP gene clusters revealed that Rhodococcus encodes many clade-specific novel RiPPs, with thirteen core peptides showing antibacterial potential. High-throughput elicitor screening (HiTES) and non-targeted metabolomics revealed that a marine-derived Rhodococcus strain produces a large number of new aurachin-like compounds when exposed to specific elicitors. The present study highlights the diversity and specificity of secondary biosynthetic potential in Rhodococcus , and provides valuable information for the targeted exploration of novel natural products from Rhodococcus , especially for phylogenomic-clade-specific metabolites.

Isoprocarb (IPC), a representative monocyclic carbamate insecticide, poses risks of environmental contamination and harm to non-target organisms. However, its degradation mechanism has not been reported. In this study, a newly IPC-degrading strain D-6 was isolated from the genus Rhodococcus, and its degradation characteristics and pathway of IPC were analyzed. A novel hydrolase IpcH, responsible for hydrolyzing IPC to 2-isopropylphenol (IPP), was identified. IpcH exhibited low similarity (< 27 %) with other reported hydrolases, including previously characterized carbamate insecticides hydrolases, indicating its novelty. The K m and k cat values of IpcH towards IPC were 69.99 ± 8.33 μM and 95.96 ± 4.02 s -1 , respectively. Also, IpcH exhibited catalytic activity towards various types of carbamate insecticides, including monocyclic carbamates (IPC, fenobucarb and propoxur), bicyclic carbamates (carbaryl and carbofuran), and linear carbamates (oxamyl and aldicarb). The molecular docking and site-directed mutagenesis revealed that His254, His256, His329 and His376 were essential for IpcH activity. Strain D-6 can effectively reduce the toxicity of IPC and IPP towards sensitive organisms through its degradation ability. This study presents the initial report on IPC degradation pathway and molecular mechanism of IPC degradation, and provides a good potential strain for bioremediating IPC and IPP-contaminated environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Two novel actinomycetal strains, designated CC-R113 T and CC-R104 T , were isolated from the tissues of two macroalgae collected on the northern Portuguese coast. Phylogenetic analyses based on the 16S rRNA gene showed that strain CT-R113 T belongs to the genus Nocardiopsis , being closely related to Nocardiopsis umidischolae 66/93 T and Nocardiopsis tropica VKM Ac-1457 T , with 98.65 and 98.39 % sequence similarity, respectively. The clade formed between the three type strains was confirmed by phylogenomic analysis. The genome of strain CT-R113 T was 7.27 Mb in size with a G+C content of 71.3 mol %, with average nucleotide identity (ANI) values of 89.59 and 90.14 % with strains 66/93 T and VKM Ac-1457 T , respectively. The major cellular fatty acids were identified as C 18 : 1  ω 9 c , iso-C 16 : 0 and anteiso-C 17 : 0 . Menaquinone 10 (MK-10) was the major respiratory quinone. Comparative analysis of 16S rRNA gene sequences showed that strain CC-R104 T belongs to the genus Rhodococcus and is most closely related to Rhodococcus pyridinivorans DSM 44555 T , with 98.24 % sequence similarity. However, phylogenomic analysis revealed that strain CC-R104 T establishes a clade with Rhodococcus artemisae DSM 45380 T , being more distant from Rhodococcus pyridinivorans DSM 44555 T . The genome of strain CC-R104 T was 5.34 Mb in size with a G+C content of 67.01 mol%. The ANI value between strains CC-R104 T and DSM 45380 T was 81.2 % and between strains CC-R104 T and DSM 44555 T was 81.5 %. The major cellular fatty acids were identified as C 18 : 1  ω 9 c , C 16 : 0 and summed feature 3. Menaquinone 8 (MK-8) was the only respiratory quinone. For both CC-R113 T and CC-R104 T , optimum growth was observed at pH 7.0, 28 °C and 0-5 % NaCl and whole-cell hydrolysates contained meso -diaminopimelic acid as the cell-wall diamino acid. On the basis of phenotypic, molecular and chemotaxonomic characteristics, strains CT-R113 T and CC-R104 T are considered to represent novel species, for which the names Nocardiopsis codii sp. nov. (type strain CT-R113 T =LMG33234 T =UCCCB172 T ) and Rhodococcus chondri sp. nov. (type strain CC-R104 T =LMG33233 T =UCCCB171 T ) are proposed.

Triclocarban (TCC) and its metabolite, 3,4-dichloroaniline (DCA), are classified as emerging organic contaminants (EOCs). Significant concerns arise from water and soil contamination with TCC and its metabolites. These concerns are especially pronounced at high concentrations of up to approximately 20 mg/kg dry weight, as observed in wastewater treatment plants (WWTPs). Here, a TCC-degrading co-culture system comprising Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 was utilized to degrade TCC (14.5 mg/L) by 85.9% in 7 days, showing improved degradation efficiency compared with monocultures. A combination of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR) was performed. Meanwhile, through the combination of further experiments involving heterologous expression and gene knockout, we proposed three TCC metabolic pathways and identified four key genes (tccG, tccS, phB, phL) involved in the TCC degradation process. Moreover, we revealed the internal labor division patterns and connections in the co-culture system, indicating that TCC hydrolysis products were exchanged between co-cultured strains. Additionally, mutualistic cooperation between BX2 and LY-1 enhances TCC degradation efficiency. Finally, phytotoxicity assays confirmed a significant reduction in the plant toxicity of TCC following synergistic degradation by two strains. The in-depth understanding of the TCC biotransformation mechanisms and microbial interactions provides useful information for elucidating the mechanism of the collaborative biodegradation of various contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida , posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus , Nocardia , and Gordonia . This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus , Gordonia , and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms., Competing Interests: The authors declare no conflict of interest.

Propanil residues can contaminate habitats where microbial degradation is predominant. In this study, an efficient propanil-degrading strain C-1 was isolated from paddy and identified as Rhodococcus sp. It can completely degrade 10 μg/L-150 mg/L propanil within 0.33-10 h via the hydrolysis of the amide bond, forming 3,4-dichloroaniline. A novel bifunctional amidase, PamC, was identified in strain C-1. PamC can catalyze the hydrolysis of the amide bond of propanil to produce 3,4-dichloroaniline as well as the hydrolysis of the ester bonds of aryloxyphenoxypropionate herbicides (APPHs, clodinafop-propargyl, cyhalofop-butyl, fenoxaprop- p -ethyl, fluazifop- p -butyl, haloxyfop- p -methyl, and quizalofop -p -ethyl) to form aryloxyphenoxypropionic acids. Molecular docking and site-directed mutagenesis confirmed that the catalytic triad Lys82-Ser157-Ser181 was the active center for PamC to hydrolyze propanil and cyhalofop-butyl. This study presents a novel bifunctional amidase with capabilities for both amide and ester bond hydrolysis and enhances our understanding of the molecular mechanisms underlying the degradation of propanil and APPHs.

Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD 600 of 0.8. OD 600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A genome of Rhodococcus rhodochrous IEGM 1362 was sequenced and annotated. This strain can transform monoterpene alcohol (-)-isopulegol with the formation of two novel pharmacologically promising metabolites. Nine genes encoding cytochrome P450, presumably involved in (-)-isopulegol transformation, were found in the genome of R. rhodochrous IEGM 1362. Primers and PCR conditions for their detection were selected. The obtained data can be used for the further investigation of genes encoding enzymes involved in monoterpene biotransformation.

Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode m ycofactocin-dependent a lcohol d egradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither Δ madA nor Δ mftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the Δ madA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2 , predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus , a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks., Competing Interests: The authors declare no conflict of interest.

Terpenoids and steroids are secondary plant and animal metabolites and are widely used to produce highly effective pharmacologically significant compounds. One of the promising approaches to the transformation of these compounds to form bioactive metabolites is their transformation using microorganisms. Rhodococcus spp. are one of the most developed objects in biotechnology due to their exceptional metabolic capabilities and resistance to extreme environmental conditions. In this review, information on the processes of biotransformation of terpenoid and steroid compounds by actinomycetes of the genus Rhodococcus and their molecular genetic bases are most fully collected and analyzed for the first time. Examples of the use of both native whole-cell catalysts and mutant strains and purified enzyme systems for the production of derivatives of terpenoids and steroids are given.

Background: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry., Methods and Results: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt., Conclusion: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Marine hydrocarbonoclastic bacteria can use polycyclic aromatic hydrocarbons as carbon and energy sources, that makes these bacteria highly attractive for bioremediation in oil-polluted waters. However, genomic and metabolic differences between species are still the subject of study to understand the evolution and strategies to degrade PAHs. This study presents Rhodococcus ruber MSA14, an isolated bacterium from marine sediments in Baja California, Mexico, which exhibits adaptability to saline environments, a high level of intrinsic pyrene tolerance (> 5 g L - 1 ), and efficient degradation of pyrene (0.2 g L - 1 ) by 30% in 27 days. Additionally, this strain demonstrates versatility by using naphthalene and phenanthrene as individual carbon sources. The genome sequencing of R. ruber MSA14 revealed a genome spanning 5.45 Mbp, a plasmid of 72 kbp, and three putative megaplasmids, lengths between 110 and 470 Kbp. The bioinformatics analysis of the R. ruber MSA14 genome revealed 56 genes that encode enzymes involved in the peripheral and central pathways of aromatic hydrocarbon catabolism, alkane, alkene, and polymer degradation. Within its genome, R. ruber MSA14 possesses genes responsible for salt tolerance and siderophore production. In addition, the genomic analysis of R. ruber MSA14 against 13 reference genomes revealed that all compared strains have at least one gene involved in the alkanes and catechol degradation pathway. Overall, physiological assays and genomic analysis suggest that R. ruber MSA14 is a new haloalkalitolerant and hydrocarbonoclastic strain toward a wide range of hydrocarbons, making it a promising candidate for in-depth characterization studies and bioremediation processes as part of a synthetic microbial consortium, as well as having a better understanding of the catabolic potential and functional diversity among the Rhodococci group., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13 T , with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13 T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13 T (= CCTCC AB 2022327 T  = JCM 35797 T ). This strain can use phenol as its sole carbon source. Z13 T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was μ max  = 0.93174 h -1 , and the maximum specific degradation rate was q max  = 0.47405 h -1 . Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13 T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13 T for soil remediation was investigated by introducing Z13 T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Atractylodes macrocephala Koidz (AMK) is a perennial herb from the plant family Asteraceae (formerly Compositae). This herb is mainly distributed in mountainous wetlands in Zhejiang, Sichuan, Yunnan, and Hunan provinces of China. Its medicinal production and quality, however, are severely impacted by root rot disease. In our previous study, endophytic bacterium designated AM201 exerted a high biocontrol effect on the root rot disease of AMK. However, the molecular mechanisms underlying this effect remain unclear. In this study, the identity of strain AM201 as Rhodococcus sp. was determined through analysis of its morphology, physiological and biochemical characteristics, as well as 16S rDNA sequencing. Subsequently, we performed transcriptome sequencing and bioinformatics analysis to compare and analyze the transcriptome profiles of root tissues from two groups: AM201 (AMK seedlings inoculated with Fusarium solani [FS] and AM201) and FS (AMK seedlings inoculated with FS alone). We also conducted morphological, physiological, biochemical, and molecular identification analyses for the AM201 strain. We obtained 1,560 differentially expressed genes, including 187 upregulated genes and 1,373 downregulated genes. We screened six key genes (GOLS2, CIPK25, ABI2, egID, PG1, and pgxB) involved in the resistance of AM201 against AMK root rot disease. These genes play a critical role in reactive oxygen species (ROS) clearance, Ca 2+ signal transduction, abscisic acid signal inhibition, plant root growth, and plant cell wall defense. The strain AM201 was identified as Rhodococcus sp. based on its morphological characteristics, physiological and biochemical properties, and 16S rDNA sequencing results. The findings of this study could enable to prevent and control root rot disease in AMK and could offer theoretical guidance for the agricultural production of other medicinal herbs., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment, intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30 °C and 10 °C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30 °C. Among them, 17β-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10 °C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain's adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30 °C and 10 °C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported., Results: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O 2 for the formation of reactive oxygen species (ROS) at different levels., Conclusions: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation., (© 2024. The Author(s).)

The genus Rhodococcus is recognized for its potential to degrade a large range of aromatic substances, including plant-derived phenolic compounds. We used comparative genomics in the context of the broader Rhodococcus pan-genome to study genomic traits of two newly described Rhodococcus strains (type-strain Rhodococcus pseudokoreensis R79 T and Rhodococcus koreensis R85) isolated from apple rhizosphere. Of particular interest was their ability to degrade phenolic compounds as part of an integrated approach to treat apple replant disease (ARD) syndrome. The pan-genome of the genus Rhodococcus based on 109 high-quality genomes was open with a small core (1.3%) consisting of genes assigned to basic cell functioning. The range of genome sizes in Rhodococcus was high, from 3.7 to 10.9 Mbp. Genomes from host-associated strains were generally smaller compared to environmental isolates which were characterized by exceptionally large genome sizes. Due to large genomic differences, we propose the reclassification of distinct groups of rhodococci like the Rhodococcus equi cluster to new genera. Taxonomic species affiliation was the most important factor in predicting genetic content and clustering of the genomes. Additionally, we found genes that discriminated between the strains based on habitat. All members of the genus Rhodococcus had at least one gene involved in the pathway for the degradation of benzoate, while biphenyl degradation was mainly restricted to strains in close phylogenetic relationships with our isolates. The ~40% of genes still unclassified in larger Rhodococcus genomes, particularly those of environmental isolates, need more research to explore the metabolic potential of this genus.IMPORTANCE Rhodococcus is a diverse, metabolically powerful genus, with high potential to adapt to different habitats due to the linear plasmids and large genome sizes. The analysis of its pan-genome allowed us to separate host-associated from environmental strains, supporting taxonomic reclassification. It was shown which genes contribute to the differentiation of the genomes based on habitat, which can possibly be used for targeted isolation and screening for desired traits. With respect to apple replant disease (ARD), our isolates showed genome traits that suggest potential for application in reducing plant-derived phenolic substances in soil, which makes them good candidates for further testing against ARD., Competing Interests: The authors declare no conflict of interest.

Background: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process., Results: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production., Conclusions: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control., (© 2024. The Author(s).)

1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To improve the titre of lignin-derived pyridine-dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid-based overexpression of seven endogenous and exogenous lignin-degrading genes was tested. Overexpression of endogenous multi-copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4-PDCA production by 2.5-, 1.4-, and 3.5-fold, respectively, while overexpression of dye-decolorizing peroxidase dypB was found to enhance titre by 1.4-fold, and overexpression of Streptomyces viridosporus laccase enhanced titre by 1.3-fold. The genomic context of the R. jostii mcoA gene suggests involvement in 4-hydroxybenzoate utilization, which was consistent with enhanced whole cell biotransformation of 4-hydroxybenzoate by R. jostii pTipQC2-mcoA. These data support the role of multi-copper oxidases in bacterial lignin degradation, and provide an opportunity to enhance titres of lignin-derived bioproducts., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The performance of bacterial strains in executing degradative functions under the coexistence of heavy metals/heavy metal-like elements and organic contaminants is understudied. In this study, we isolated a fluorene-degrading bacterium, highly arsenic-resistant, designated as strain 2021, from contaminated soil at the abandoned site of an old coking plant. It was identified as a member of the genus Rhodococcus sp. strain 2021 exhibited efficient fluorene-degrading ability under optimal conditions of 400 mg/L fluorene, 30 °C, pH 7.0, and 250 mg/L trivalent arsenic. It was noted that the addition of arsenic could promote the growth of strain 2021 and improve the degradation of fluorene - a phenomenon that has not been described yet. The results further indicated that strain 2021 can oxidize As 3+ to As 5+ ; here, approximately 13.1% of As 3+ was converted to As 5+ after aerobic cultivation for 8 days at 30 °C. The addition of arsenic could greatly up-regulate the expression of arsR/A/B/C/D and pcaG/H gene clusters involved in arsenic resistance and aromatic hydrocarbon degradation; it also aided in maintaining the continuously high expression of cstA that codes for carbon starvation protein and prmA/B that codes for monooxygenase. These results suggest that strain 2021 holds great potential for the bioremediation of environments contaminated by a combination of arsenic and polycyclic aromatic hydrocarbons. This study provides new insights into the interactions among microbes, as well as inorganic and organic pollutants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microorganism lipid droplet small regulator (MLDSR) is a transcriptional regulator of the major lipid droplet (LD)-associated protein MLDS in Rhodococcus jostii RHA1 and Rhodococcus opacus PD630. In this study, we investigated the role of MLDSR on lipid metabolism and triacylglycerol (TAG) accumulation in R. jostii RHA1 at physiological and molecular levels. MLDSR gene deletion promoted a significant decrease of TAG accumulation, whereas inhibition of de novo fatty acid biosynthesis by the addition of cerulenin significantly repressed the expression of the mldsr-mlds cluster under nitrogen-limiting conditions. In vitro and in vivo approaches revealed that MLDSR-DNA binding is inhibited by fatty acids and acyl-CoA residues through changes in the oligomeric or conformational state of the protein. RNAseq analysis indicated that MLDSR not only controls the expression of its own gene cluster but also of several genes involved in central, lipid, and redox metabolism, among others. We also identified putative MLDSR-binding sites on the upstream regions of genes coding for lipid catabolic enzymes and validated them by EMSA assays. Overexpression of mldsr gene under nitrogen-rich conditions promoted an increase of TAG accumulation, and further cell lysis with TAG release to the culture medium. Our results suggested that MLDSR is a fatty acid-responsive regulator that plays a dual role in cells by repression or activation of several metabolic genes in R. jostii RHA1. MLDSR seems to play an important role in the fine-tuning regulation of TAG accumulation, LD formation, and cellular lipid homeostasis, contributing to the oleaginous phenotype of R. jostii RHA1 and R. opacus PD630., (© 2023 Federation of European Biochemical Societies.)

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p -methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p -methoxybenzoate ( p -MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p -MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p -MBA and several derivatives. Furthermore, a deletion mutant of a candidate p- hydroxybenzoate ( p -HBA) hydroxylase, Δ pobA , did not grow on p -HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O -demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a Δ pcaL strain grew on neither p -MBA nor veratrate, indicating they are catabolized through the β-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus , a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives., Competing Interests: The authors declare no conflict of interest.

Rhodococcus equi is an opportunistic pathogen known to cause pulmonary and extrapulmonary disease among immunocompromised patients. Treatment is frequently challenging due to intrinsic resistance to multiple antibiotic classes. While non- equi Rhodococcus spp. are prevalent, their clinical significance is poorly defined. There is also limited data on antibiotic susceptibility testing (AST) of Rhodococcus infection in humans. We conducted a single-center, retrospective cohort study evaluating clinical characteristics, microbiologic profile, and AST of Rhodococcus infections between June 2012 and 2022 at our tertiary academic medical center. Identification of Rhodococcus spp. was performed by Sanger 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry, and AST was performed by agar dilution. Three hundred twenty-two isolates of Rhodococcus spp. were identified from blood (50%), pulmonary (26%), and bone/joint (12%) sources. R. equi/hoagii, R. corynebacterioides , and R. erythropolis were the most frequently isolated species, with 19% of isolates identified only to genus level. One hundred ninety-nine isolates evaluated for AST demonstrated high-level resistance to amoxicillin/clavulanate, cephalosporins, and aminoglycosides. More than 95% susceptibility to imipenem, vancomycin, linezolid, rifampin, and clarithromycin was observed. Non- equi species showed a significantly more favorable AST profile relative to R. equi . Clinically significant Rhodococcus infection was rare with 10 cases diagnosed (majority due to R. equi ) and managed. The majority of patients received 2- or 3-drug combination therapy for 2-6 months, with favorable clinical response. Significant differences in AST were observed between R. equi and non- equi species. Despite high antimicrobial resistance to several antibiotic classes, imipenem and vancomycin remain appropriate empiric treatment options for R. equi . Future research evaluating mechanisms underlying antimicrobial resistance is warranted., Competing Interests: The authors declare no conflict of interest.

Rhodococci have been regarded as ideal chassis for biotransformation, biodegradation, and biosynthesis for their unique environmental persistence and robustness. However, most species of Rhodococcus are still difficult to metabolically engineer due to the lack of genetic tools and techniques. In this study, synthetic sRNA strategy was exploited for gene repression in R. erythropolis XP. The synthetic sRNA based on the RhlS scaffold from Pseudomonas aeruginosa functions better in repressing sfgfp expression than those based on E. coli MicC, SgrS, and P. aeruginosa PrrF1-2 scaffold. The RhlS-based sRNAs were applied to study the influence of sulfur metabolism on biodesulfurization (BDS) efficiency in R. erythropolis XP and successfully identified two genes involved in sulfur metabolism that affect the BDS efficiency significantly. The RhlS-based synthetic sRNAs show promise in the metabolic engineering of Rhodococcus and promote the industrial applications of Rhodococcus in environmental remediation and biosynthesis., (© 2024 Wiley‐VCH GmbH.)

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Strains belonging to R. opacus, R. jostii, R. fascians, R. erythropolis and R. equi exhibited differential ability to grow and produce lipids from fruit residues (grape marc and apple pomace), as well as single carbohydrates, such as glucose, gluconate, fructose and sucrose. The oleaginous species, R. opacus (strains PD630 and MR22) and R. jostii RHA1, produced higher yields of biomass (5.1-5.6 g L -1 ) and lipids (38-44% of CDW) from apple juice wastes, in comparison to R. erythropolis DSM43060, R. fascians F7 and R. equi ATCC6939 (4.1-4.3 g L -1 and less than 10% CDW of lipids). The production of cellular biomass and lipids were also higher in R. opacus and R. jostii (6.8-7.2 g L -1 and 33.9-36.5% of CDW of lipids) compared to R. erythropolis, R. fascians, and R. equi (3.0-3.6 g L -1 and less than 10% CDW of lipids), during cultivation of cells on wine grape waste. A genome-wide bioinformatic analysis of rhodococci indicated that oleaginous species possess a complete set of genes/proteins necessary for the efficient utilization of carbohydrates, whereas genomes from non-oleaginous rhodococcal strains lack relevant genes coding for transporters and/or enzymes for the uptake, catabolism and assimilation of carbohydrates, such as gntP, glcP, edd, eda, among others. Results of this study highlight the potential use of the oleaginous rhodococcal species to convert sugar-rich agro-industrial wastes, such as apple pomace and grape marc, into single-cell oils., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O 2 ) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components., (Copyright © 2024. Published by Elsevier Inc.)

Polyethylene (PE), a widely used recalcitrant synthetic polymer, is a major global pollutant. PE has very low biodegradability due to its rigid C-C backbone and high hydrophobicity. Although microorganisms have been suggested to possess PE-degrading enzymes, our understanding of the PE biodegradation process and its overall applicability is still lacking. In the present study, we used an artificial bacterial consortium for PE biodegradation to compensate for the enzyme availability and metabolic capabilities of individual bacterial strains. Consortium members were selected based on available literature and preliminary screening for PE-degrading enzymes, including laccases, lipases, esterases, and alkane hydroxylases. PE pellets were incubated with the consortium for 200 days. A next-generation sequencing ana-lysis of the consortium community of the culture broth and on the PE pellet identified Rhodococcus as the dominant bacteria. Among the Rhodococcus strains in the consortium, Rhodococcus erythropolis was predominant. Scanning electron microscopy (SEM) revealed multilayered biofilms with bacteria embedded on the PE surface. SEM micrographs of PE pellets after biofilm removal showed bacterial pitting and surface deterioration. Multicellular biofilm structures and surface biodeterioration were observed in an incubation of PE pellets with R. erythropolis alone. The present study demonstrated that PE may be biodegraded by an artificially constructed bacterial consortium, in which R. erythropolis has emerged as an important player. The results showing the robust colonization of hydrophobic PE by R. erythropolis and that it naturally possesses and extracellularly expresses several target enzymes suggest its potential as a host for further improved PE biodeterioration by genetic engineering technology using a well-studied host-vector system.

Steroidal estrogens residues in the environment can be a serious hazard to humans and animals and has been listed as group 1 carcinogens by World Health Organization (WHO). Microbial degradation is one of the effective strategies for the removal of such contaminants. In this study, a low-temperature degrading bacterial strain (Rhodococcus sp. RCBS9) was isolated from the soil of a dairy farm for 17β-estradiol (E2) degradation. The strain RCBS9 exhibited an efficient degradation potential at low temperatures. To lean how different factors influence E2 degradation, we have found a major role of intracellular enzymes in E2 degradation. Genomic and metabolomic analyses have suggested potential degradation genes and four metabolic pathways. These findings provide valuable strain resources for the low temperature bioremediation of E2 contamination and insights into E2 biodegradation mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus' metabolic pathways converting them to lipids are analyzed from a "beginning-to-end" view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus' utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. The possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Unleashing the Capacity of Rhodococcus for Converting Lignin into Lipids”., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians are phytobacteria that induce crown gall and leafy gall disease, respectively, resulting in undesirable growth abnormalities. When present in nurseries, plants infected by either bacterium are destroyed, resulting in substantial losses for growers, especially those producing plants valued for their ornamental attributes. There are many unanswered questions regarding pathogen transmission on tools used to take cuttings for propagation and whether products used for bacterial disease control are effective. We investigated the ability to transmit pathogenic A. tumefaciens and R. fascians on secateurs and the efficacy of registered control products against both bacteria in vitro and in vivo . Experimental plants used were Rosa × hybrida , Leucanthemum × superbum , and Chrysanthemum × grandiflorum for A. tumefaciens and Petunia × hybrida and Oenothera 'Siskiyou' with R . fascians. In separate experiments, we found secateurs could convey both bacteria in numbers sufficient to initiate disease in a host-dependent manner and that bacteria could be recovered from secateurs after a single cut through an infected stem. In in vivo assays, none of six products tested against A. tumefaciens prevented crown gall disease, although several products appeared promising in in vitro trials. Likewise, four compounds trialed against R . fascians failed to prevent disease. Sanitation and clean planting material remain the primary means of disease management., Competing Interests: The author(s) declare no conflict of interest.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoA R , which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoA R and its partner protein gcoB R resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoA R and gcoB R restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoA R could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl- O -demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O -demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O -demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O -demethylase in vitro . This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization., Competing Interests: The authors declare no conflict of interest.

Microbial desulfurization has been extensively studied as a promising alternative to the widely applied chemical desulfurization process. Sulfur removal from petroleum and its products becomes essential, as the environmental regulations become increasingly stringent. Rhodococcus qingshengii IGTS8 has gained ground as a naturally occurring model biocatalyst, due to its superior specific activity for desulfurization of dibenzothiophene (DBT). Recalcitrant organic sulfur compounds-DBT included-are preferentially removed by selective carbon-sulfur bond cleavage to avoid a reduction in the calorific value of the fuel. The process, however, still has not reached economically sustainable levels, as certain limitations have been identified. One of those bottlenecks is the repression of catalytic activity caused by ubiquitous sulfur sources such as inorganic sulfate, methionine, or cysteine. Herein, we report an optimized culture medium for wild-type stain IGTS8 that completely alleviates the sulfate-mediated repression of biodesulfurization activity without modification of the natural biocatalyst. Medium C not only promotes growth in the presence of several sulfur sources, including DBT, but also enhances biodesulfurization of resting cells grown in the presence of up to 5 mM sulfate. Based on the above, the present work can be considered as a step towards the development of a more viable commercial biodesulfurization process., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Rhodococcus bacteria are a fast-growing platform for biocatalysis, biodegradation, and biosynthesis, but not a platform for molecular biology. That is, Rhodococcus are not convenient for genetic engineering. One major issue for the engineering of Rhodococcus is the absence of a publicly available, curated, and commented collection of sequences of genetic parts that are functional in biotechnologically relevant species of Rhodococcus ( R. erythropolis , R. rhodochrous , R. ruber , and R. jostii ). Here, we present a collection of genetic parts for Rhodococcus (vector replicons, promoter regions, regulators, markers, and reporters) supported by a thorough analysis of their functionality. We also highlight and discuss the gaps in Rhodococcus -related genetic parts and techniques, which should be filled in order to make these bacteria a full-fledged molecular biology platform independent of Escherichia coli . We conclude that all major types of required genetic parts for Rhodococcus are available now, except multicopy replicons. As for model Rhodococcus strains, there is a particular shortage of strains with high electrocompetence levels and strains designed for solving specific genetic engineering tasks. We suggest that these obstacles are surmountable in the near future due to an intensification of research work in the field of genetic techniques for non-conventional bacteria.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Phthalate diesters are extensively used as plasticizers in manufacturing plastic materials; however, because of their estrogenic properties, these chemicals have emerged as a global threat to human health. The present study investigated the course of degradation of a widely used plasticizer, benzyl butyl phthalate (BBP), by the bacterium PAE-6, belonging to the genus Rhodococcus . The metabolism of BBP, possessing structurally dissimilar side chains, was evaluated biochemically using a combination of respirometric, chromatographic, enzymatic, and mass-spectrometric analyses, depicting pathways of degradation. Consequently, the biochemical observations were corroborated by identifying possible catabolic genes from whole-genome analysis, and the involvement of inducible specific esterases and other degradative enzymes was validated by transcriptomic, reverse transcription-quantitative PCR (RT-qPCR) and proteomic analyses. Nonetheless, phthalic acid (PA), an intermediate of BBP, could not be efficiently metabolized by strain PAE-6, although the genome contains a PA-degrading gene cluster. This deficiency of complete degradation of BBP by strain PAE-6 was effectively managed by using a coculture of strains PAE-6 and PAE-2. The latter was identified as a Paenarthrobacter strain which can efficiently utilize PA. Based on sequence analysis of the PA-degrading gene cluster in strain PAE-6, it appeared that the alpha subunit of the multicomponent phthalate 3,4-dioxygenase harbors a number of altered residues in the multiple sequence alignment of homologous subunits, which may play a role(s) in poor turnover of PA. IMPORTANCE Benzyl butyl phthalate (BBP), an estrogenic, high-molecular-weight phthalic acid diester, is an extensively used plasticizer throughout the world. Due to its structural rigidity and hydrophobic nature, BBP gets adsorbed on sediments and largely escapes the biotic and abiotic degradative processes of the ecosystem. In the present study, a potent BBP-degrading bacterial strain belonging to the genus Rhodococcus was isolated that can also assimilate a number of other phthalate diesters of environmental concern. Various biochemical and multi-omics analyses revealed that the strain harbors all the required catabolic machinery for the degradation of the plasticizer and elucidated the inducible regulation of the associated catabolic genes and gene clusters., Competing Interests: The authors declare no conflict of interest.

Rhodococcus opacus is a bacterium that has a high tolerance to aromatic compounds and can produce significant amounts of triacylglycerol (TAG). Here, we present iGR1773, the first genome-scale model (GSM) of R. opacus PD630 metabolism based on its genomic sequence and associated data. The model includes 1773 genes, 3025 reactions, and 1956 metabolites, was developed in a reproducible manner using CarveMe, and was evaluated through Metabolic Model tests (MEMOTE). We combine the model with two Constraint-Based Reconstruction and Analysis (COBRA) methods that use transcriptomics data to predict growth rates and fluxes: E-Flux2 and SPOT (Simplified Pearson Correlation with Transcriptomic data). Growth rates are best predicted by E-Flux2. Flux profiles are more accurately predicted by E-Flux2 than flux balance analysis (FBA) and parsimonious FBA (pFBA), when compared to 44 central carbon fluxes measured by 13 C-Metabolic Flux Analysis ( 13 C-MFA). Under glucose-fed conditions, E-Flux2 presents an R 2 value of 0.54, while predictions based on pFBA had an inferior R 2 of 0.28. We attribute this improved performance to the extra activity information provided by the transcriptomics data. For phenol-fed metabolism, in which the substrate first enters the TCA cycle, E-Flux2's flux predictions display a high R 2 of 0.96 while pFBA showed an R 2 of 0.93. We also show that glucose metabolism and phenol metabolism function with similar relative ATP maintenance costs. These findings demonstrate that iGR1773 can help the metabolic engineering community predict aromatic substrate utilization patterns and perform computational strain design.

As a crucial growth factor, thiamine can regulate functional microbial communities; however, our current understanding of its effect on bioremediation is lacking. Using metatranscriptome and 16S rRNA gene sequencing, we explored the mechanism of response of an efficient tetrahydrofuran (THF)-degrading microbial culture, designated H-1, to exogenous thiamine. Rhodococcus ruber ZM07, a strain performing the THF degradation function in H-1, is a thiamine-auxotrophic bacterium. Furthermore, thiamine affected the microbial community structure of H-1 by altering resource and niche distributions. A microbial co-occurrence network was constructed to help us identify and isolate the cooperators of strain ZM07 in the microbial community. Based on the prediction of the network, two non-THF-degrading bacteria, Hydrogenophaga intermedia ZM11 and Pigmentiphaga daeguensis ZM12, were isolated. Our results suggest that strain ZM11 is a good cooperator of ZM07, and it might be more competitive than other cooperators (e.g., ZM12) in cocultured systems. Additionally, two dominant strains in our microbial culture displayed a "seesaw" pattern, and they showed completely different responses to exogenous thiamine. The growth of the THF degrader ZM07 was spurred by additional thiamine (with an increased relative abundance and significant upregulation of most metabolic pathways), while the growth of the cooperator ZM11 was obviously suppressed under the same circumstances. This relationship was the opposite without thiamine addition. Our study reveals that exogenous thiamine can affect the interaction patterns between THF- and non-THF-degrading microorganisms and provides new insight into the effects of micronutrients on the environmental microbial community. IMPORTANCE Auxotrophic microorganisms play important roles in the biodegradation of pollutants in nature. Exploring the interspecies relationship between auxotrophic THF-degrading bacteria and other microbes is helpful for the efficient utilization of auxotrophic functional microorganisms. Herein, the thiamine-auxotrophic THF-degrading bacterium ZM07 was isolated from the microbial culture H-1, and the effect of thiamine on the structure of H-1 during THF bioremediation was studied. Thiamine may help ZM07 occupy more niches and utilize more resources, thus improving THF degradation efficiency. This research provides a new strategy to improve the THF or other xenobiotic compound biodegradation performance of auxotrophic functional microorganisms/microbial communities by artificially adding special micronutrients. Additionally, the "seesaw" relationship between the thiamine-auxotrophic strain ZM07 and its prototrophic cooperator ZM11 during THF bioremediation could be changed by exogenous thiamine. This study reveals the effect of micronutrients on microbial interactions and provides an effective way to regulate the pollutant biodegradation efficiency of microbial communities., Competing Interests: The authors declare no conflict of interest.

Due to low consumption and high efficiency, in situ microbial remediation of petroleum hydrocarbons (PHs)-contaminated sites in in-service petrochemical enterprises has attracted more and more attention. In this study, a degrading strain was isolated from oil depot-contaminated soil with soil extract (PHs) as the sole carbon source, identified and named Rhodococcus sp. OBD-3. Strain OBD-3 exhibited wide adaptability and degradability over a wide range of temperatures (15-37 °C), pH (6.0-9.0), and salinities (1-7% NaCl) to degrade 60.6-86.6% of PHs. Under extreme conditions (15 °C and 3-7% salinity), PHs were degraded by 60.6 ± 8.2% and more than 82.1% respectively. In OBD-3, the alkane monooxygenase genes alkB1 and alkB2 (GenBank accession numbers: MZ688386 and MZ688387) were found, which belonged to Rhodococcus by sequence alignment. Moreover, strain OBD-3 was used in lab scale remediation in which the contaminated soil with OBD-3 was isolated as the remediation object. The PHs were removed at 2,809 ± 597 mg/kg within 2 months, and the relative abundances of Sphingobium and Pseudomonas in soil increased more than fivefold. This study not only established a system for the isolation and identification of indigenous degrading strains that could efficiently degrade pollutants in the isolated environment but also enabled the isolated degrading strains to have potential application prospects in the in situ bioremediation of PHs-contaminated soils., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

3-Methylindole (skatole) is regarded as one of the most offensive compounds in odor emission. Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic. In this study, an efficient aerobic skatole-degrading consortium was obtained. Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses. Bioinformatic analyses indicated that the skatole downstream degradation was mainly via the catechol pathway, and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase. Genome binning and gene analyses indicated that Pseudomonas, Pseudoclavibacter, and Raineyella should cooperate with Rhodococcus for the skatole degradation process. Moreover, a pure strain Rhodococcus sp. DMU1 was successfully obtained which could utilize skatole as the sole carbon source. Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium. Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway. Collectively, our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader, and the degradation mainly proceeded via the catechol pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The chloroxylenol (PCMX) degrading strain was successfully isolated from sludge and identified as Rhodococcus ruber (R. ruber). Afterwards, a bioaugmentation system was constructed by seeding R. ruber into nitrifying sludge to fasten degradation efficiency of highly toxic PCMX from wastewater. Results showed that R. ruber presented high PCMX-degrading performance under aerobic conditions, 25 °C, pH 7.0 and inoculum sizes of 4% (v/v). These optimized conditions were used in subsequent bioaugmentation experiment. In bioaugmentation system, R. ruber could detoxify nitrifiers by degrading PCMX, and the content of polysaccharide in extracellular polymeric substances increased. The quantitative polymerase chain reaction results exhibited that the absolute abundance of 16S rRNA gene and ammonia oxidizing bacteria (AOB) slightly elevated in bioaugmentation system. After analyzing the results of high-throughput sequencing, it was found that the loaded R. ruber can colonize successfully and turn into dominant strains in sludge system. Molecular docking simulation showed that PCMX had a weaker suppressed effect on AOB than nitrite oxidizing bacteria, and R. ruber can alleviate the adverse effect. This study could provide a novel strategy for potential application in reinforcement of PCMX removal in wastewater treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Biodegradation of pyridine pollutant by microorganisms is one of the economical and effective methods to solve the environmental pollution of pyridine under high salinity conditions. To this end, screening of microorganisms with pyridine degradation capability and high salinity tolerance is an important prerequisite. In this paper, a salt-resistant pyridine degradation bacterium was isolated from the activated sludge of Shanxi coking wastewater treatment plant, and identified as a bacterium belonging to Rhodococcus on the basis of colony morphology and 16S rDNA gene phylogenetic analysis. Salt tolerance experiment showed that strain LV4 could grow and degrade pyridine with the initial concentration of 500 mg/L completely in 0%-6% saline environment. However, when the salinity was higher than 4%, strain LV4 grew slowly and the degradation time of pyridine by strain LV4 was significantly prolonged. Scanning electron microscopy showed that the cell division of strain LV4 became slower, and more granular extracellular polymeric substance (EPS) was induced to secrete in high salinity environment. When the salinity was not higher than 4%, strain LV4 responded to the high salinity environment mainly through increasing the protein content in EPS. The optimum conditions for pyridine degradation by strain LV4 at 4% salinity were 30 ℃, pH 7.0 and 120 r/min (DO 10.30 mg/L). Under these optimal conditions, strain LV4 could completely degrade pyridine with an initial concentration of 500 mg/L at a maximum rate of (29.10±0.18) mg/(L·h) after 12 h adaptation period, and the total organic carbon (TOC) removal efficiency reached 88.36%, indicating that stain LV4 has a good mineralization effect on pyridine. By analyzing the intermediate products in pyridine degradation process, it was speculated that strain LV4 achieved pyridine ring opening and degradation mainly through two metabolic pathways: pyridine-ring hydroxylation and pyridine-ring hydrogenation. The rapid degradation of pyridine by strain LV4 in high salinity environment indicates its application potential in the pollution control of high salinity pyridine environment.

Organosulfur compounds in fossil fuels have been a major concern in the process of achieving zero-sulfur fuel production. Biodesulfurization (BDS) is an environmentally friendly strategy for the removal of refractory organosulfur compounds from fossil fuels. Even though researchers are committed to engineering the desulfurization-specific pathway for improving BDS efficiency, the industrial application of BDS is still difficult. Recently, the sulfur metabolism of Rhodococcus has begun to attract attention due to its influences on the BDS process. In this review, we introduce the sulfur metabolism in Rhodococcus, including sulfur absorption, reduction, and assimilation; and summarize desulfurization in Rhodococcus, including the desulfurization mechanism, the regulation mechanism of the 4S pathway, and the strategies of optimizing the 4S pathway to improve BDS efficiency. In particular, the influence of sulfur metabolism on BDS efficiency is discussed. In addition, we consider the latest genetic engineering strategies in Rhodococcus. An improved understanding of the relationship between sulfur metabolism and desulfurization will enable the industrial application of BDS., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Carotenoids are a series of natural pigments with unique structure and physiological functions. In this study, a novel Rhodococcus aetherivorans strain N1 was discovered, which can produce 6.4 mg/g carotenoids including β-carotene, zeaxanthin and isorenieratene from glucose. Moreover, strain N1 can directly produce 3.0 mg/g carotenoids from the undetoxified straw hydrolysate, representing the highest carotenoids production from the undetoxified lignocellulosic hydrolysate. The crude carotenoid extracts of strain N1 showed efficient free radical scavenging activity and stability. Strain N1 has complete methylerythritol 4-phosphate (MEP) pathway and related genes for carotenoid synthesis, especially the rare aromatic carotenoid of isorenieratene. Genomic comparison between strain N1 and other carotenoid producing Rhodococcus sp. strains showed the conservatism and universality of carotenoids synthesis gene. These results proved that R. aetherivorans strain N1 can serve as a promising producer for the industrialization of carotenoid production., Competing Interests: Declaration of Competing Interest Authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nitrile hydratase (NHase, EC 4.2.1.84) is an excellent biocatalyst that catalyzes the hydration of nitrile substances to their corresponding amides. Given its catalytic specificity and eco-friendliness, NHase has extensive applications in the chemical, pharmaceutical, and cosmetic industries. To improve the affinity between Rhodococcus erythropolis CCM2595-derived NHase (ReNHase) and adiponitrile, this study used a semirational design to improve the efficiency of ReNHase in catalyzing the generation of 5-cyanopentanamide from adiponitrile. Enzyme kinetics analysis showed that Km of the mutant ReNHase B:G196Y was 3.265 mmol l -1 , which was lower than that of the wild-type NHase. The affinity of the mutant ReNHase B:G196Y to adiponitrile was increased by 36.35%, and the efficiency of the mutant ReNHase B:G196Y in catalyzing adiponitrile to 5-cyanopentamide was increased by 10.11%. The analysis of the enzyme-substrate interaction showed that the hydrogen bond length of the mutant ReNHase B:G196Y to adiponitrile was shortened by 0.59 Å, which enhanced the interaction between the mutant and adiponitrile and, thereby, increased the substrate affinity. Similarly, the structural analysis showed that the amino acid flexibility near the mutation site of ReNHase B:G196Y was increased, which enhanced the binding force between the enzyme and adiponitrile. Our work may provide a new theoretical basis for the modification of substrate affinity of NHase and increase the possibility of industrial applications of the enzyme.

Rhodococcus is a genus of actinomycetes that has been explored by the scientific community for different purposes, especially for bioremediation uses. However, the mechanisms governing Rhodococcus-mediated bioremediation processes are far from being fully elucidated. In this sense, this work aimed to compile the recent advances in the use of Rhodococcus for the bioremediation of organic and inorganic contaminants present in different environmental compartments. We reviewed the bioremediation capacity and mechanisms of Rhodococcus spp. in the treatment of polycyclic aromatic hydrocarbons, phenolic substances, emerging contaminants, heavy metals, and dyes given their human health risks and environmental concern. Different bioremediation techniques were discussed, including experimental conditions, treatment efficiencies, mechanisms, and degradation pathways. The use of Rhodococcus strains in the bioremediation of several compounds is a promising approach due to their features, primarily the presence of appropriate enzyme systems, which result in high decontamination efficiencies; but that vary according to experimental conditions. Besides, the genus Rhodococcus contains a small number of opportunistic species and pathogens, representing an advantage from the point of view of safety. Advances in analytical detection techniques and Molecular Biology have been collaborating to improve the understanding of the mechanisms and pathways involved in bioremediation processes. In the context of using Rhodococcus spp. as bioremediation agents, there is a need for more studies that 1) evaluate the role of these actinomycetes on a pilot and field scale; 2) use genetic engineering tools and consortia with other microorganisms to improve the bioremediation efficiency; and 3) isolate new Rhodococcus strains from environments with extreme and/or contaminated conditions aiming to explore their adaptive capabilities for bioremediation purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria., (© 2022. The Author(s).)

Lignin represents the most abundant renewable aromatics in nature, which has complicated and heterogeneous structure. The rapid development of biotransformation technology has brought new opportunities to achieve the complete lignin valorization. Especially, Rhodococcus sp. possesses excellent capabilities to metabolize aromatic hydrocarbons degraded from lignin. Furthermore, it can convert these toxic compounds into high value added bioproducts, such as microbial lipids, polyhydroxyalkanoate and carotenoid et al. Accordingly, this review will discuss the potentials of Rhodococcus sp. as a cell factory for lignin biotransformation, including phenol tolerance, lignin depolymerization and lignin-derived aromatic hydrocarbon metabolism. The detailed metabolic mechanism for lignin biotransformation and bioproducts spectrum of Rhodococcus sp. will be comprehensively discussed. The available molecular tools for the conversion of lignin by Rhodococcus sp. will be reviewed, and the possible direction for lignin biotransformation in the future will also be proposed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The widespread use of plastic has led to the global occurrence of phthalate esters (PAEs) pollution. PAEs can be effectively removed from polluted environments by microbe-mediated degradation. Di-(2-ethylhexyl) phthalate (DEHP) has the highest residual concentration in agricultural soil-contaminated areas compared to other PAEs in most of China. The Rhodococcus pyridinovorans DNHP-S2 microbial isolate identified was found to efficiently degrade DEHP. Within a 72 h period, the bacteria were able to degrade 52.47% and 99.75% of 500 mg L -1 DEHP at 10 °C and 35 °C, respectively. Dimethyl phthalate (DMP) was first identified as an intermediate metabolite of DEHP, which is different from the previously reported DEHP catabolic pathway. Genomic sequencing of DNHP-S2 identified benzoate 1,2-dioxygenase and catechol 2,3/1,2-dioxygenase as potential mediators of DEHP degradation, consistent with the existence of two downstream metabolic pathways governing DEHP degradation. Three targets DEHP metabolism-related enzymes were found to be DEHP-inducible at the mRNA level, and DNHP-S2 was able to mediate the complete degradation of DEHP at lower temperatures, as confirmed via RT-qPCR. DNHP-S2 was also found to readily break down other PAEs including DMP, di-n-butyl phthalate (DBP), di-n-octyl phthalate (DnOP), and n-butyl benzyl phthalate (BBP). Together, these results thus highlight DNHP-S2 as a bacterial strain with great promise as a tool for the remediation of PAE pollution. In addition to providing new germplasm and genetic resources for use in the context of PAE degradation, these results also offer new insight into the potential mechanisms whereby PAEs undergo catabolic degradation, making them well-suited for use in PAE-contaminated environments., (Copyright © 2022 Elsevier Ltd. All rights reserved.)